This project investigates signal transduction pathways that regulate proliferation, migration, and differentiation of lens and corneal epithelial cells with the goal of identifying enzymes that may be therapeutically targeted in pathological conditions. The primary concentration in this fiscal year has been to determine the role of the proline-directed kinase, Cdk5, in corneal epithelial cell wound healing. Studies that were in progress last fiscal year to determine the relationship between Cdk5 and Src-family kinases in regulating corneal epithelial cell migration were completed in this year. These studies established that Src-family kinases are necessary for corneal cell migration and are responsible for activation of Cdk5. Cdk5,in turn, retards the transport of activated Src from the perinuclear region to peripheral cell sites and decreases the total amount of activated Src in the cell. We further demonstrated that Cdk5 regulates transport of Src to the leading edge of a corneal debridement wound in the mouse corneal epithelium in an organ culture system or the leading edge of a scratch wound in cultured mouse corneal epithelial cells. The relationship between Src and Cdk5 is being extended by site-directed mutagenesis of potential phosphorylation sites on both kinases, in conjunction with functional and biochemical studies. The ongoing collaboration with Dr. Mary Ann Stepp (GWU) has begun preliminary tests of the safety and efficacy of Cdk5 inhibitors in promoting corneal wound closure in mice. We are also exploring the role of Cdk5 in the lens. Since our previous work implicated Cdk5 in both cell-to-matrix and cell-to-cell adhesion in this tissue, we have investigated possible mechanisms that may mediate these effects. One avenue of investigation has been to identify lens proteins that interact with Cdk5 or its activating proteins, p35 and p39, using the yeast two-hybrid technique. These studies have identified two proteins, muskelin and myosin light chain, as specific interactors of p39. Both of these proteins are involved in cytoskeletal regulation, and may be important for Cdk5-dependent regulation of cell-matrix or cell-cell adhesion. In parallel studies, we constructed transgenic mice that overexpress Cdk5 or a kinase inactive form of Cdk5 only in lens fibers under the direction of the betaB1 crystallin promoter. Analysis of these animals suggests that Cdk5 promotes cell-cell adhesion of lens fibers. Although this result is at odds with other findings indicating that Cdk5 has the opposite effect on cell-cell adhesion, it is consistent with effects we have observed in the corneal epithelium. We are continuing our collaboration with Dr. Sue Menko, (Thomas Jefferson University) to reconcile these discrepant findings and to determine the role or roles of Cdk5 in modulating cell-cell adhesion. Present studies are focused on the relationship between N-cadherin turnover and Cdk5.